Who really discovered trisomy 21? (righting an old wrong)

A few days ago a French student in my Useful Genetics Coursera course posted a link to an article in Le Monde (sorry, it's both in French and behind a paywall, but this link might get you a translation).  It reported that a Jan. 31 award ceremony for the discovery of the cause of Down syndrome, part of the 7th Human and Medical Genetics Congress  in Bordeaux, had been blocked by a Down syndrome support organization (Fondation Jerome-Lejeune).  The back story is very depressing, an egregious example of a woman scientist being denied credit for her discovery.

Photo source: Le Monde

The woman is Dr. Marthe Gautier, now 88 years old.  In 1956 she was a young physician, returning to Paris from a year's study of pediatric cardiology at Harvard.  She was given a clinical/teaching position at a local hospital, with no funds for research.  The Head of the Pediatric Unit, Raymond Turpin, was interested in mongolism (as Down syndrome was then called); years earlier he had proposed that it might be caused by a chromosome abnormality.  Human cytogenetics was not well understood, but a big breakthrough came this same year, when the true chromosome number was finally established as 46 (not 48).  When Turpin complained that nobody was investigating his hypothesis, Gautier proposed that she take this problem on, since her Harvard training had introduced her to both cell culture and histology.  Turpin agreed to provide a tissue sample from a patient.

For this work she was given a disused laboratory with a fridge, a centrifuge, and a poor quality microscope, but no funding.  And of course she still had her other responsibilities.  But she was keen and resourceful, so she took out a personal loan to buy glassware, kept a live cockerel as a source of serum, and used her own blood when she needed human serum.

By the end of 1957 she had everything working with normal human cells, and could clearly distinguish the 46 chromosomes.  So she asked Prof. Turpin for the patient sample.  After 6 months wait it arrived, and she quickly was able to prepare slides showing that it had not 46 but 47 chromosomes, with three copies of a small chromosome.  But her microscope was very poor, and she could not identify the chromosome or take the photographs of her slides that a publication would need.

All this time Prof. Turpin had never visited her lab, but she'd had frequent visits from a protege of his, Jerome Lejeune.  When she showed Lejeune her discovery, he offered to take the slides to another laboratory where they could be photographed.  She never saw the slides again, but the photographs appeared in Montreal two months later (August 1958), where Lejeune announced to the International Conference of Human Genetics in Montreal that he had discovered the cause of Down syndrome!  Lejeune and Turpin quickly wrote up 'their' discovery, with Gautier as middle author, but Gautier only learned about this publication the day before it appeared in print.

These were tough times for a woman scientist in France, and Gautier decided not to fight for the credit for her discovery, instead returning to her clinical and teaching work on congenital heart diseases.

Lejeune became not just a renowned researcher but the darling of the French Catholic right-to-life movement.  You can read long flattering Wikipedia biographies in both French and English. He was showered with awards and given a prestigious Chair of Human Genetics at the Paris School of Medicine, bypassing the usual competition.

When prenatal diagnosis became available Lejeune campaigned against it on religious grounds. He became a friend of Pope John Paul II and was appointed President of the Pontifical Academy for Life (Wikipedia), the Catholic think-tank for medical ethics.  He died in 1994.  The Fondation Jerome-Lejeune was established in his honour; there's an American branch too.  This foundation provides funds for research into Down syndrome and support for families and patients, but only in the context of very strong opposition to abortion.  They're also campaigning to have Lejeune beatified by the Vatican.

But Gautier's role in the discovery of trisomy 21 was not totally forgotten.  It has been very well described in a 2009 article in the journal Human Genetics by Gautier and Peter Harper, the author of a major history of cytogenetics (paywalled but try this link to a pdf), and in a 2013 interview.  There's also a French Wikipedia page about her.  But few people in the field know about this injustice, and cytogenetics textbooks and courses still credit Lejeune for the discovery.  Gautier has no English Wikipedia page, and the Wikipedia pages on Lejeune describe her contribution as follows:

"Using a new tissue culture technique brought back from the United States by his colleague Marthe Gautier, Lejeune began working with her to count the number of chromosomes in children with Down syndrome. The laboratory notebook begun by Dr. Lejeune on July 10, 1957 indicates that on May 22, 1958, he succeeded in showing, for the first time, the presence of 47 chromosomes in a child with Down syndrome."

Not surprisingly, the Fondation Jerome-Lejeune strongly opposes any correction of the scientific record, since this would reveal the intellectual theft at the base of their hero's reputation.  That's why they sent in the bailiffs to record her award ceremony.

I'm of course outraged to learn about this situation, and this post is one attempt to set the record straight.  My other venue is Wikipedia, which I've been learning to edit.  So far I've added sentences crediting Gautier with the discovery to the Wikipedia entries on Down Syndrome and on Jerome Lejeune.  Someone else had added a mention of the dispute to his French entry.  I'm going to expand these each into a paragraph.  I've also created an empty page for Marthe Gautier and requested that the Wikipedia translation people fill it from her French entry.

So please spread the word.  Marthe Gautier discovered that trisomy 21 is the cause of Down syndrome, and Jerome Lejeune's saintly reputation is based on scientific fraud.

Later:  I've corrected an error: Gautier could not identify the trisomic chromosome with her poor microscope.  And here are links to news articles about this controversy, in Nature and Science.

Plans for RNA-seq analyses

I should have posted this after last week's lab meeting but am only now getting to it.  I sensibly took snapshots of the whiteboard at the end of that lab meeting, so I could check what we'd decided.

The issues:  We have several Haemophilus influenzae mutants whose gene-expression profiles we want to examine, either during competence development in the MIV starvation medium or during normal growth in the rich culture medium sBHI.  For most of these (i) we want samples from several timepoints over a few hours, (ii)we want wildtype controls done in the same experiment, and (iii) we want three replicate samples from experiments done on different days.  And it would be nice to have multiples of 24 samples, since the kits and sequencing are most efficient with that.

That's a lot of constraints , but we came up with a plan that meets them all:  The first two days are the samples that have already been prepared and sequenced; the other 6 days are for me to generate the samples.

 

The samples will consist of viable cells frozen in glycerol (one or two 1.5 ml tubes), and duplicate pellets of cells that have been briefly incubated with a RNA-protection reagent to stabilize their RNA.  Later the frozen viable cells will be thawed and transformed to check that they have the expected level of competence - I can probably do all of them in a few days.

After all the samples have been collected, the RNA-prep pellets will be thawed and the RNA isolated using a kit.  After a quick check of RNA concentration and size, the contaminating DNA is removed by treatment with Turbo-DNase.

All the samples are then checked for concentration and quality using a special something (the post-doc recommends, using equipment in another lab), then treated to remove the bulk of the ribosomal RNA (this kit costs a lot, I think $200 per sample), then rechecked for concentration and quality using the special something.

Finally the samples are ready to be made into multiplexed sequencing libraries (expensive service on campus) and then sequenced (another service on campus).

To order (first check what we have on hand):

•  RNA protection reagent for 72 samples
• RNA prep kits for 72 samples
• Turbo-DNase for 72 samples
• Does the 'special something' require special reagents?
• rRNA-removing kits for 72 samples

The reasons for doing these experiments are described briefly in this post.  

Because my hydroxyurea experiments found that cell doubling is substantially faster when cultures are at very low cell density, for the first samples in experiments F. G and H I'll use large volumes of cells at a lower cell density than the usual OD600=0.2.  Getting the right density will be a bit tricky since we can't use OD600 to check densities of very dilute cultures.  In the table above I said I'd use an OD600 of 0.02, but I'll start by growing cells from a lower density to about this OD and then diluting them 16-fold so they'll have another four doublings before I sample them.  The hydroxyurea-experiment cultures were much more dilute than this, but I need to balance culture density and culture volume so I'll have enough cells for the RNA extractions.  With this plan I'll be collecting cells from about 2 x 250 ml of culture for the first samples, and combining the remaining volumes in the two flasks for the later samples.

Other experiments to do:  We need to sequence the genome of strain RR753, to identify candidate mutations that cause its hypercompetence.  Before doing this I should recheck it and the backcross strain I made - if they both are hypercompetent we'll want to sequence both.

But it's MY figure!

The postdoc and I have a minireview coming out in an ASM journal, and we're at the 'permissions' stage.

One of the figures is an explanatory diagram I drew for this article (figure on the left below).  It's similar to an explanatory diagram I drew for an article we published in Genetics a few years ago (on the right below).  Because one of the reviewers asked if our minireview figure was adapted from the published figure, in the Figure Legend we wrote '(adapted from (91)' even though it wasn't. 
.

Now the Production Editor is asking us for the formal permission from Genetics to republish this figure. I explained that it wasn't really adapted from the published one, but because the figures are similar she recommends that we request permission.

OK.....

So Genetics has a 'Get Permissions' link beside each article, and that sends me to the Copyright Clearance Center, where I specify that I want permission to republish their content in a journal.  I click on Price & Order (not a good sign). 

That takes me to another form, asking me questions I don't understand ('Duration of use'?  'Lifetime unit quantity'?).  Whatever.

I click on 'Get Price'.

I click on 'Continue'.  This takes me to a 'Login' page.

 Of course I don't have an account.  I create an account.

Now I log in with my account, but I don't see any record that they remember what I was asking for.  So I do it all again.

Several more pages.

 I click 'Add to Cart'.

My order is not complete until I click 'Checkout'.

My order is not complete until I accept the Terms and Conditions.

OK, my order is complete.  They're sending me an email confirmation.  They say it could take 15 days....

(Added later:  In fact it took less than 24 hr!)

On the other hand, I  just also requested permission to use a figure from our Nucleic Acids Research paper.  Very quick and easy - permission granted immediately.

List of RNAseq analyses to do

In case we have money for lots of RNAseq runs, here's a list of every sample that might be useful:

24 samples = 1 run:
Replicates of the samples we've already done:  MIV competence induction at t=0, t=10, t=30 and t=100:

• 1 x KW20, 
• 1 x sxy-, 
• 2 x ∆HI0659, 
• 2 x ∆HI0660.

Miscellaneous samples (3 replicates of each?):

• KW20 in late-log (time of max 'spontaneous' competence)
• KW20 in log phase + hydroxyurea: t=0, t=30? t=60? 
• HI0659/0660 double mutant, in MIV at t=0 (= log phase) and t=???
• murE749 mutant in log phase growth and stationary phase
• Other hypercompetence mutants:  murE750, 751, 752?  in log phase growth
• mystery hypercompetence mutant RR753 or backcross, in log phase and late-log?
• KW20 in log phase growth as control (duplicating earlier MIV t=0?)
• gcvA mutant in MIV?  or in murE749 background in log phase?
• ∆hfq mutant in MIV?  in log phase too.
• purR? purH, crp? cya?
• Cells in MIV + AMP.

What I've done lately

('Lately' being the 20 months since I last updated the Table of Contents of my lab notebook.)

I've been keeping a Table of Contents of my lab notebooks since I was a grad student, initially on paper but for the past 20+ years as a Word file. As can be seen from the screenshot above, each experiment has a number, and I record the date and a few words about what I was trying to do and what I found. Before I kept this blog, searching it was the easy way to find experiments on a particular problem, and it's still a very valuable resource.

One good thing about keeping a Table of Contents is that updating it forces me to go back over every experiment I've done lately.  So I've just done that for everything since May 2012, and it was very informative.  Here I'm going to write a summary of the experiments that I'd like to now follow up on, especially noting where RNAseq would be appropriate.

Experiments with the HI0659/0660 antitoxin/toxin genes:  We had found that a HI05659 knockout mutation completely prevents competence, that a HI0660 mutation doesn't affect competence, and that both genes were homologous to a known antitoxin/toxin pair.  This suggested that HI0660 encodes a toxin that blocks competence and HI0659 encodes an antitoxin that blocks this.  Now I've shown: Competence is not restored by the sxy1 or murE749 hypercompetence mutations, which act by increasing sxy expression. The HI0659 mutation does not cause a dramatic change in expression of lacZ fusions to the competence genes comA and rec2.  A HI0659/0660 double mutant transforms normally, confirming the hypothesis that HI0659's job is to block the activity of HI0660.  The HI0659 mutant grows just like wildtype in a BioScreen culture, suggesting that the toxin is either not expressed in noncompetent cells or has no activity that affects growth.  Gene expression in these mutants has been examined by RNAseq.  I'll describe these results in another post, but replication is needed.

Experiments with the hypercompetence mutation murE749 and peptidoglygan recycling mutations:  I first remade all the strains and rechecked the MIV-induced competence phenotypes of our set of four peptidoglygan recycling mutants, in a wildtype background with and without added cyclic AMP and in sxy1 and murE749 backgrounds.  There was quite a bit of variation, but all were approximately normal on replication.  The occasional dramatic differences may just be noise, or could result from some sensitivity to the details of the experiments.  The only consistent difference is that the gcvA mutation (a putative regulator) was about 3-5-fold less competent than the others.  We should definitely do RNAseq of the murE749 mutant, and maybe of the gcvA mutant too.

Phenotype of a hfq mutant:  hfq encodes an RNA-binding protein that interacts with many regulatory small RNAs, so we and others have hypothesized that it plays a role in regulating the translatability of sxy mRNA.  The RA made a knockout, which I found has normal growth but a consistent 10-fold reduction in transformation in late-log, in MIV and in a sxy1 hypercompetent background.  The drop was more extreme in an overnight culture - this should be retested. This mutant is a good candidate for RNAseq analysis

Effect of hydroxyurea:  The small molecule hydroxyurea (HU) specifically inhibits the enzyme ribonucleotide reductase, which converts NTPs (RNA precursors) to dNTPs (DNA precursors).  Thus HU depletes dNTP pools and stalls replication forks (demonstrated in E. coli).  If H. influenzae competence is a response to blockage of DNA replication fork progression, HU should induce competence.  But it doesn't.  If competence protects H. influenzae cells from the harmful effects of stalled replication forks, competent cells should be less sensitive to HU.  But they aren't: cells that become transformed show the same sensitivity to HU as non-competent cells, and mutations that cause hypercompetence do not reduce sensitivity to growth arrest or killing by HU.

BUT. these is a connection between competence and HU.  One competence-induced gene turns out to provide substantial protection against the harmful effects of HU.  dprA is in all characterized competence regulons, where it coats incoming DNA and promotes homologous recombination.  it's also present in many more species not know to ever become competent, although no non-competence function is known.  A dprA knockout has about the same effect on HU sensitivity as a mutation in recBC, which is well established as serving mainly to help DNA replication recover from stalling.  But an E. coli dprA mutant isn't more sensitive to HU.

Isolating more hypercompetent mutants:  Highly desirable, but a badly executed series of experiments.  It did produce lots of EMS-mutagenized cells stored at -80°C.  I should first test these for induced novR mutations, and if seen then redo the selection for hypercompetent mutants.   These mutants, if obtained, would be good candidates for RNAseq analysis.

Planning more RNAseq experiments

The postdoc and former RA generated some great RNAseq data, which I'll write about in another post.  But we have some money that needs to be spent on sequencing in the next couple of months, so we need to decide which additional RNA seq runs we should do.  And then I'm going to grow the cultures and prep the RNAs.

We have data sets showing how RNA levels change after transfer to competence-inducing MIV medium for several Haemophilus influenzae strains: wildtype (2 expts), sxy- (2 expts), HI0659- (1 (antitoxin?, 1 expt) and HI0660- (toxin?, 1 expt).  For each we have samples at t=0, t=10, t=30 and t=100 minutes.  (The figure shows a comparison between wildtype and sxy- at t=0 and t=10; the red circles are CRP-regulated genes and the blue ones are competence genes.)  We need to do at least one replicate of the HI0659 and HI0660 cultures.  If we also did another replicate of everything, that would be a full 24 sample run (one lane?) for the sequencer, and enough data that we could do proper statistical analyses.

But I also want to get RNAseq data for strains with other mutations, especially the hypercompetence-causing mutation murE749 in exponential growth.  This would be a single condition, replicated once or twice, so 2 or 3 samples total.  I might be able to squeeze this in with the run described above; depending on what other experiments we plan to do with these strains, two replicates of some might be enough.  Or it might be better to do a second run since we have the funds, doing a more comprehensive analysis of other conditions and other mutants too.

One condition I'd like to examine is 'late-log' growth, where wildtype cells develop moderate levels of competence.  I want to see if these levels are comparable to those in the HI0659 antitoxin mutant, which shows no competence at all although it appears to have (compared to wildtype cells) only a slight decrease in competence gene expression at t=10, no decrease at t=30, and a moderate decrease at t=100.  It's possible that the toxin acts only by decreasing mRNA levels of other competence genes, but the disproportion between its absolute competence defect and modest RNA defect makes me wonder if it also does something else.

We have three other hypercompetence mutants with mutations in murE - I don't know if it would be worth doing one sample of each of these in exponential growth.  We also have a hypercompetent mutant of unknown genotype - RNAseq might find the mutation as well as show us the RNA changes.  Might it be worth testing a crp mutant or cya mutant, to confirm our understanding of cAMP/CRP regulation?  Or the purR or purH mutant, or cells whose MIV-induced competence development has been blocked by adding AMP?  Or cells whose DNA replication has been blocked by hydroxyurea (depletes dNTP pools).  Or the hfq mutant, which has 10-fold lower competence.

Research direction and goals (pondering)

Depending on when I count from, I've spent the past twenty or thirty years trying to get people to think rigorously about whether bacteria have any processes that evolved to promote random recombination of chromosomal alleles or genes.

And I've largely failed at this.  A few people think my ideas are reasonable, but the great majority of microbiologists and evolutionary biologists continue to comfortably assume that genetic exchange happens in bacteria because it's an evolutionary good thing.  I'm still pretty sure they're wrong, but I think I've done almost all I can to change their minds.

I'm at a decision point.  The Canadian Institutes of Health Research (CIHR) has funded the bulk of my work, but recent proposals have failed and I don't have any compelling projects to propose.  The funding environment has moved on, leaving little support for 'pure' research, especially where others think your perspective is wrongheaded. So I don't think I'm going to submit a proposal, at least not this year.

I'm not going to stop doing research.  Nor am I going to shift from benchwork to bioinformatics, at least not yet and not all the way.  I have good research space and facilities, a small pure-science grant that will support my new grad student, and funds remaining in my previous CIHR grant that can probably be extended for another year.  And I have lots of projects, lots of questions I want to answer about H. influenzae competence.

So my tentative plan is to relax a bit (i.e. not write that damned CIHR proposal) and get back in the lab.  I'm also teaching two courses this term, so I certainly won't be slacking off, but I see no point in putting this extra pressure on myself yet again.

An important paper about the interactions between influenza and H. influenzae infection

Wong SM, Bernui M, Shen H, & Akerley BJ (2013). Genome-wide fitness profiling reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung. Proceedings of the National Academy of Sciences of the United States of America, 110 (38), 15413-8 PMID: 24003154

Haemophilus influenzae is a bacterium that causes respiratory tract diseases, but it's often confused with the virus that causes influenza (also a respiratory tract disease).  The modern confusion arises from the similarity of the names, but the name similarity arises from an older confusion about the cause of influenza.

The influenza virus wasn't identified until after the big 'Spanish influenza' pandemic at the end of World War I.   At the time of the epidemic the cause of influenza was still being sought, and a small heme-requiring bacterium informally called 'Pfeiffer's influenza bacillus' (now Haemophilus influenzae) was a likely suspect, since it was found in the lungs of many influenza victims.  We now know that influenza itself is caused by a family of small RNA-genome viruses, and that H. influenzae commonly causes a secondary pneumonia, especially in people whose respiratory tracts have been weakened by other diseases or by old age.

A recent paper from Brian Akerley's group set out to identify the bacterial genes that contribute to this effect.  Their hypothesis was that some H. influenzae functions that are needed for normal infection (in the absence of influenza virus) would not be needed when the virus was present, i.e. that the virus infection allows H. influenzae to take shortcuts.

Their strategy was to infect mouse lungs with a mixed population of H. influenzae mutants carrying transposon insertions in many different genes, and then examine which mutations become lost during the infection.  Cells that have mutations in genes that don't matter during infection will do just fine, but those with mutations in genes needed during infection will be unable to grow and so their DNA will be missing from the final population.  These experiments compared a healthy population of mice with mice that had been lightly infected with influenza A virus 5 days before the H. influenzae infection.  These authors had previously examined genes needed for single infection (Gawronski et al. 2009), and another group (Lee et al. 2010) had shown a few years previously that preinfection of mice with influenza increased the severity of H. influenzae infection, but this new work used a much less virulent strain of H. influenzae.

About 30% of the H. influenzae genes were excluded from the analysis, most because they were either essential or contributed to growth in lab culture medium, and some because they were duplicated or too small to analyze. This left about 1200 genes whose roles in infection and coinfection could be analyzed.

Most of these genes were found to not be important for either type of infection.  (The researchers' criteria for 'important' were quite stringent, so this doesn't mean they make no contribution.)  But 85 genes were required for both types of infection, with another 24 required only in single infection and another 18 required only in the coinfection with influenza virus.

The infection after influenza virus is probably a good model for similar human infections.  But the significance of the simple infection is less clear.  Since the bacteria are quickly cleared from the lung, it's not clear what's being evaluated.  There's also the possibility of chance effects playing a big role here, which might explain why the genes identified by this experiment are not very consistent with those found by a very similar experiment reported by this lab a few years ago (Gawronsky et al 2009).

I went through this paper in the hope that it would give us evidence of the in vivo importance of competence genes, but it doesn't.  None of the competence-inducible (CRP-S regulated) genes are important for the conditions the authors investigated.  In fact, knockouts of quite of few of them are enriched in the recovered lung samples, suggesting that these genes may do more harm than good.

I'll write a separate post considering where my research should be going.

Back to blogging

I haven't posted anything in ages, partly because I haven't done an experiment in ages.  I'm not planning any experiments right now, but I do need to do a lot of thinking and writing about research because it's once again grant proposal time.  March 3 is the deadline for CIHR Operating Grant proposals.

I've been struggling to get started, mainly because I couldn't identify an angle that reviewers would find persuasive. The more I learn about grant writing the more I realize how important it is to get the reviewer excited about your proposal; enthusiastic reviewers will overlook problems that bored reviewers would jump on.  However a discussion today with the postdoc has suggested an angle that might work well - it's at least good enough to get going with.

Topic:  In vivo role of the Haemophilus influenzae CRP-S regulon 

Background:  This regulon controls a wide range of phenotypes important for pathogenesis: biofilm formation, motility, DNA uptake, gene transfer, DNA replication...).  It's active in vivo.  We know a lot (but not enough) about its in vitro regulation.  We've made and phenotyped knockouts of every gene. 

Goals:  Develop genetic tools for in vivo studies of the H. influenzae CRP-S regulon (e.g. control and inducible m-cherry fusions).  Use in vitro and simulated-RT studies to characterize the effects of known respiratory tract conditions on regulon expression. In collaboration with an established animal-research lab (chinchilla ear or mouse lung model), carry out in vivo investigations of regulon expression and effects on pathogenesis.. one of the established animal models.  

This angle builds very well on all our work under the previous grant (we're running on a no-cost extension so I guess it still counts as our current grant) and we can submit this proposal as a renewal rather than as a new grant.

One thing we'll need to do is identify a collaborator in whose lab the in vivo work can be done.  Sending a senior grad student or postdoc to such a lab will be much more cost effective than setting up our own facilities.

Thinking about the fraction-competent issues

The post-doc is polishing up has latest manuscript, and on reading it over yesterday I realized that we have data to clarify a very old issue.  Here's a link to an old post on this topic: http://rrresearch.fieldofscience.com/2011/05/fraction-competent-problem.html

In the old days, the best way to estimate the distribution of competence among the cells of a 'competent' culture was to measure the proportion of cells that became transformed by selectable markers that were on two separate DNA fragments (usually markers carried by a single donor strain, but far enough apart on the chromosome that they were never taken up on the same fragment.

Such assays typically find that cells that have been transformed by one marker are enriched for cells transformed by the other marker - the fraction of double transformants is higher than expected from the fractions of either single transformants - and this is used to estimate the fraction of the cells in the culture that are not competent.

Now, from the post-doc's work, we have data telling us what fractions of the cells have acquired one selected marker have also acquired one, two, three or more unselected fragments of donor DNA, and we want to use this data to unpack the relationship above.

IF:
1. all parts of the donor chromosome are equally likely to be taken up and recombined into the recipient chromosome,
and
2. all the cells in the competent culture are equally likely to take up DNA
and
3. taking up and recombining one DNA fragment does not alter the cell's probability of taking up and recombining another one,
and
4. we ignore that only a single strand is replaced at a recombination site, and that the donor and recipient strands at this site separate the first time the cell divides
and
5. we ignore the possibility of mismatch repair at these heteroduplex sites.

THEN:
the frequency of cells transformed by two unlinked markers should be the product of the frequencies of cells transformed by each marker (under assumption 1 above, equal to the square of the single-marker transformation frequency).

For now I'm mainly interested in what happens if assumptions 2 and 3 are not valid.  (The post-doc is more interested in 4 and 5, and the plan (hope) is that we'll pool our thinking and explain the whole business.)

Most traditional analysis has focused on relaxing assumption 2.  If some cells in the culture are equally competent and the rest not competent at all (and all the other assumptions hold), then the fraction of the cells that are competent is the ratio of the product of the two observed single transformation frequencies to the observed double-transformation frequency.  If the ratio is 1.0, then we'd conclude that all the cells are competent. 

What if we instead relax assumption 3?  What if taking up one DNA fragment uses up cellular resources and so reduces the cells' ability to take up more fragments?  At the extreme, no cell would take up more than one fragment.  The frequency of cotransformation would then be zero, and our ratio would be infinity.  If the resource-depletion was not absolute, then the ratio would be smaller, but it would still be larger than 1.0.  So if we observed  ratio greater than 1.0, we'd conclude that some cells can only take up one DNA fragment.

But deviations from assumptions 2 and 3 push the ratio in opposite directions.  The typical observation of a ratio less than 1.0 means that assumption 2 must be invalid but not that assumption 3 is valid. In principle, how far below 1.0 the ratio is sets a limit on how much of an effect deviations from assumption 3 can be having, but we haven't worked out the math to calculate this.  For example, if the observed ratio were 0.5, could this be because a quarter of the cells were competent but only half of those were able to take up more than one fragment?

Conversely, seeing a ratio greater than 1.0 would mean that assumption 3 was invalid but not that assumption 2 was valid, and we could in principle calculate the range of deviations consistent with a particular observed ratio. 

We could also view relaxing assumptions 2 and 3 a different way, thinking about variation in the levels of expression of competence genes leading to differences in how much machinery/resources cells have available.  What if some cells are more competent than others?  Some cells might not have enough resources to take up even a single fragment, some might have only enough resources to take up a single fragment, and some might have enough resources to take up two or more fragments.  What would our ratio look like then?

So.  For each of two selected markers, the post-doc's data gives us the actual distributions of cells that also took up no, one, two or three additional fragments: 0.35 had none, 0.40 had one, 0.21 had two, and 0.04 had three.  What can we do with these numbers?

The single-segment transformation frequency for the two selected markers was each about 0.03.  I forget the double-transformant frequency, but I remember that it predicted that only about 0.1 of the cells were competent (relaxing assumption 2), so I think it must have been about 0.01.

How far could assumption 3 be relaxed and still give the observed numbers?  Could 0.2 of the cells have been competent if only 0.65 of these were able to take up more than one fragment?


Consistent with this, only two of 20 unselected clones had recombined donor segments. 

If I have lots of KanR colonies from yesterday's hypercompetence-enrichment step...

Today:

1. Pool the colonies (from each of 6 original mutagenized cultures).
2. Freeze part.
3. Make chromosomal DNA preps from the rest.  These will be used for sequencing and for the backcrosses (steps 5 & 6)
4. As controls also make DNA from the pooled NovR colonies from yesterday.
5. Use the 2 DNAs from the StrR parent and the 2 DNAs from the CmR parent to transform KW20 to StrR and to CmR.
6. Maybe use the DNAs from the wildtype parent to transform KW20 to KanR.
7. (Incidentals: make lots more BHI agar, pour plates, use frozen competent KW20, test transformation by NalR fragment, wash and autoclave flasks again.)

Tomorrow (one final round of hypercompetence selection):

1. Pool the backcross colonies derived from each of 6 original mutagenized cultures.
2. Freeze part.
3. Grow part in log phase for at least 2 hr (like yesterday)
4. Transform with a different marker (NalR fragment, or MAP7 DNA with selection for NalR or SpcR).

Monday:

1. Pool the transformant colonies (from each of 6 original mutagenized cultures).
2. Make chromosomal DNA preps from them for sequencing.

Yet more on-the-fly experiment planning

Hm, yesterday's attempt to do two rounds of enrichment for hypercompetent mutants yielded very few colonies (about 10 total).  That's probably because I didn't let the NovR cells grow to a high enough density before transforming them with KanR DNA.  There were so few cells present with the KanR DNA was added, 5 x 10^5/ml - 6 x 10^6/ml based on the numbers of colonies that grew on the plain plates after the second round transformation).

But I can do the second round enrichment again, by pooling all the NovR colonies that grew on these plain plates.  I have between 1000 and 60,000 colonies to pool, depending on the culture, and the cultures were diluted so much that I don't have to worry that any of these colonies might be KanR. And the colonies are all NovR transformants from the previous round, though many of them are identical descendants of the original mutants.

Plan:  Put some BHI on the plates and scrape them to resuspend all the colonies.  Dilute the pooled cells in sBHI to OD600 = 0.1, then dilute 50-fold in more sBHI.  Grow (37°C, shaking) for 2-3 hr, until OD600 reaches 0.05.  Add KanR chromosomal DNA and let continue growing for another hr.  Dilute and plate on plain, Nov and Kan plates.

Progress:  OK, I've counted the colonies (previously had just estimated them), pooled them (excluding one contaminated plate), and now they're growing in 10 ml sBHI at a starting OD600 of 0.002.  Now I need to pour lots of plates.

AAACCCKKK!!! - I screwed up the selection

I did Day 1 of the big experiment yesterday.  But because the NovR DNA fragment the postdoc gave m hadn't transformed very well in the test, and I didn't have enough of it for the large volumes I was transforming, I used MAP7 DNA for the transformations that select for hypercompetent mutants.  At the time I thought the only disadvantages were a slightly lower overall transformation frequency and the 1-2% risk (for each new mutant) that transformation would also replace the mutation itself.

I forgot that, because the MAP7 DNA carries resistance to all the antibiotics we commonly select for, the pool of NovR cells I'd get would contain 1-2% cells resistant to teach of the other antibiotics.  This means that I can't just reselect the cells in the NovR pool for hypercompetence using DNA with another mutation, because this will just select those cells in the pool that got that mutation in the first round of selection for hypercompetence.

I see several options:

Option 1:  Yesterday I froze lots of mutagenized cells that hadn't yet been incubated with any DNA.  I can thaw these out, grow them for an hour at low density, and transform them to NovR with either the NovR DNA fragment or chromosomal NovR DNA that doesn't carry any other antibiotic resistances.  (I may have an old stock of this DNA.)   Then I can grow the pooled cells in medium with novobiocin for 6 hr, dilute them, and do the second-round transformation (NalR or KanR DNA)) and plate.  If I'm using the NovR fragment I may need to do this transformation on cells that have been concentrated - this will increase background, but the next round of hypercompetence-selection should take care of this.

Option 2 (BAD): I can plate the NovR transformants and manually check single colonies for resistance to the antibiotic I want to use next, by toothpicking them onto other antibiotic plates.  Then I could either test them individually for hypercompetence or pool the ones that are sensitive to whatever antibiotic I plan to use next.  Testing them individually would make the planned pool-sequencing unnecessary, and pooling them would be a lot of work.  But I might as well freeze my pools of NovR cells for later manual screening, just in case we  want to do this.

Option 3 (BAD):  I can make DNA from the pooled NovR colonies from the strain carrying the StrR mutation linked to sxy and the strain carrying the CmR mutation lnked to murE, and use this DNA to transform wildtype cells to StrR and CmR. Then I can do another round of hypercompetence selection, transforming the pooled cells with a marker that's not linked to the one I selected for.  BUT, these new pools will probably (maybe) include cells with the unselected antibiotic resistances from the MAP7 DNA.

Expanded plans for Option 1: 

1. Thaw 1 vial of each of the 7 cultures (wildtype treated with 0, 0.05 and 0.08 mM EMS, StrR and CmR treated with 0.05 and 0.08 mM EMS).  
2. Dilute way down in sBHI, to a density equivalaent to that used yesterday.  Grow 1 hr or more at low density.  
3. Look for NovR chromosomal DNA.  If using the PCR fragment, concentrate the cells by filtration just before adding the DNA.
4. Incubate cells with DNA for 15 or 30 min, then DNase-I-treat, filter, wash, and resuspend in sBHI + Novobiocin.  
5. Grow 6 hr or more, keeping OD600 below 0.1.  
6. Transform a fraction of this culture (no benefit from using it all).  Add KanR chromosomal DNA or NalR PCR fragment.  Incubate 15 min, DNase-treat and plate (if KanR) or grow for 90 min before plating (if NalR).

Timing for the big mutagenesis experiment

In the previous planning post I ended with the following breakdown:

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

- See more at: http://rrresearch.fieldofscience.com/#sthash.0AuGwsEC.dpuf

Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow,
            freeze, dilute and grow, transform with NovR, wash,
            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

 I'd like to get the whole thing one as quickly as possible, because next week may be our last chance until next April to get the DNAs in for sequencing.

I have the EMS, and I've streaked out the strains, but I didn't inoculate single colonies last night.  Could I still do the big Day 1 mutagenesis, transformation and selection today, or will there not be enough time to have the cells grow up first from single colonies?

What's the actual time commitment for day 1?

1. Inoculate cells from single colonies into sBHI, grow to OD600 = 0.1.  (time = several hr)
2. Add equal volume of sBHI containing 2X the desired concentrations of EMS.  Incubate 30 min at 37°C.  Cool cells down quickly. (time = 30 min)
3. Filter-wash cultures (7 cultures) and resuspend in larger volumes of sBHI. (time = 30 min?)
4. Grow washed cells for 90 min or until OD is back to 0.1. (time = 90 min)
5. Dilute part of each culture and grow for 1 hr longer. (time = 1 hr)
6. While cells are growing, filter-concentrate and freeze the rest of the cultures.
7. Add NovR DNA fragment to cultures. Incubate 30 min, then DNase I for 5 min. (time = 40 min)
8. Filter cultures, resuspend in sBHI + novobiocin. (time = 30 min)
9. Grow (at least 6 hr or) overnight.
10. The next morning, plate some cells on Nov5 plates, freeze some, and grow some back into log phase for KanR transformation with MAP7 DNA.

OK, I think I can do steps 1-8 today.

How much EMS to use:

In #181 I calculated that adding 79.5 µl of EMS to 15 ml culture gave 0.05 mM, and 127.3 µl gave 0.08 mM.  So for my 10 ml of cells I would use 53 µl and 85 µl of EMS.

What about the volumes of culture to use?

1. Mutagenize 10 ml at OD600 = 0.1
2. Resuspend in 80 ml.
3. Freeze 70 ml after 90 min (concentrate cells first) and dilute 10 ml to 40 ml in fresh sBHI
4. Grow 1 hr, add DNA etc, filter.
5. Freeze half the cells.
6. Resuspend the rest in 100 ml + novobiocin2.5.  Grow 6 hr - overnight

Should I mutagenize a larger volume?  10 ml will be only about 3 x 10^9 cells.  In #181 (the original EMS experiment) I used 15 ml at OD600 of 0.33, which is about 5 times as many cells.

How much EMS do I have?  OK, 5 ml (after brief "Where on earth did I put it???").  It doesn't keep well once opened ("Store under inert gas") so maybe I should double the volumes for the first two steps and freeze more of the mutagenized cells for possible later analysis.  So use 106 µl and 170 µl EMS for 20ml cultures, and dilute to 160 ml.

What about the EMS-contaminated waste culture and tips?

EMS is inactivated in 1.0 M NaOH.  My filter flask will contain about 400 ml of EMS waste (including washes), so I'll add 16 g of solid NaOH to that, and let it sit for an hour before neutralization and disposal.  I'll put the contaminated tips in there too.

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

- See more at: http://rrresearch.fieldofscience.com/#sthash.dxAGHC0t.dpuf

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

- See more at: http://rrresearch.fieldofscience.com/#sthash.0AuGwsEC.dpuf

Timing of competence development in a new murE749 transformant

In the previous post I realized that I don't know how quickly competence will develop in cells that have just acquired a murE hypercompetence mutation.  In that post this was a practical problem (how long should the mutagenized cells be incubated for), but it also has research-grade implications.  That's because we still have no idea how these mutations turn on the competence regulon.  If they take a long time to have an effect, I would suspect some sort of gradually developing imbalance that eventually changes Sxy expression, whereas if they act quickly I'd suspect a more direct effect.

How could I find this out?  I would need to introduce the murE749 hypercompetence mutation into a murE+ cell, presumably by transformation, and then follow the development of competence.  I can do this because we have strains with a CmR cassette that's about 70% linked to murE in transformations. Strain RR797 has the cassette and the murE749 mutation; strain RR805 has the cassette and murE+. So if I transform competent wildtype cells with RR797 DNA and select for chloramphenicol resistance, about 70% of the CmR cells should have the murE749 mutation.

If I do this CmR selection in broth (plating on the side just to check that the transformation worked), I can check how long it takes for the culture to increase its log-phase competence from a transformation frequency of <10 10="" 70="" a="" about="" expected="" i="" mixture="" of="" s="" that="" the="" to="">murE749
.  In parallel, as a control that will also provide useful information, I'll transform with RR805 DNA and measure how long the CmR cells and unselected cells take to completely lose competence and resume exponential growth in sBHI at low density.  (I think I've measured loss of competence before but not carefully.)
How long will this take?  I think it's a one-day experiment after I make the DNAs (or if I find old stocks of the DNAs in my fridge (later - found RR805 DNA but not RR797 DNA so I'll plan on remaking them both).

1. Pour lots of plain, Cm and Cm+Nov plates
2. Thaw out frozen competent Rd cells
3. Incubate with DNAs, add DNase I for 5 min
4. Filter to wash away the DNA and (more importantly) the DNase I
5. Resuspend in sBHI (t = 0)
6. Grow 1 hr, add chloramphenicol (1 µg/ml I think)
7. Take aliquots to tubes with MAP7 DNA at t = 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr (longer?).  
8. Dilute and plate on plain, Cm and Cm+Nov plates.

Would we learn anything?  Yes if competence development after transformation is very fast (appears as quickly as chloramphenicol resistance) or very slow, no otherwise.

The EMS mutagenesis mutant hunt - progress and plans

I wrote about this general experimental plan here, and described the preliminary test of the NovR selection here.  The EMS mutagen has been ordered and should arrive within the next few days.  The experiment isn't as urgent as we originally thought, because the DNA sequencing plans have changed, but it still needs to be done soon, so I'd better be ready to get started.  Here's the plan:

Do the initial mutagenesis in three different strains:

1. Wildtype H. influenzae Rd
2. H. influenzae Rd carrying the normal sxy gene and a streptomycin-resistance mutation.  StrR is only about 50 kb from sxy, so we can use it to enrich for hypercompetence mutations in sxy.
3. H. influenzae Rd carrying the normal murE gene and a chloramphenicol-resistance mutation about 4 kb from murE, which we can use it enrich for hypercompetence mutations in murE.

To minimize spontaneous competence the cells will be pre-grown in sBHI at low density for at least 2 hr.

Each strain will be incubated with 0.0, 0.05 and 0.08 mM EMS for 30 min at an initial OD600 of 0.05. Maybe the volume will be 10 ml.

After the 30 min mutagenesis each culture will be thoroughly washed by filtration to remove the EMS and resuspended in fresh sBHI (50 ml? 100 ml?). 

First plan: The cultures will be grown for 2 hr - that should be enough for any mutational changes in sxy to cause elevated competence.  The density will need to be kept low - ideally below OD600 = 0.1.  Will it be enough time for expression of the murE-mutant hypercompetence phenotype?  Hard to say, since we don't know how the mutations cause their phenotype.  Ideally I would do an experiment to find this out.  (How? Not easily, it seems...   I could transform competent wildtype cells with DNA from a CmR murE749 mutant, selecting for CmR after 1 hr expression time, and then at intervals (every hour?)  transforming aliquots of the culture to NovR with MAP7 DNA.  But the cells would initially have to be competent, so I'd have to grow the culture for at least a couple of hours at low density to grow-out this competence before I would be able to detect any log-phase competence caused by the murE mutation...)  Instead I think I'll just hope that 2 hr of log-phase growth is enough time for a new murE mutation (or any other mutation) to cause hypercompetence.  Wait, is there any compelling reason not to grow them longer?  The volume will keep getting larger with the repeated dilutions, but I could just freeze some of the cells after 1 or 2 hr and continue growing the rest for another hour or two.  The frequency of the mutants I'm looking for shouldn't change with growth, since none of the hypercompetence mutations we know of slow growth.

So new plan:  Grow the mutagenized cultures for 90 minutes.  The OD should be back up to about 0.1.  Freeze 3/4 of the cells (concentrate by filtration before freezing).  Dilute the rest back to OD 0.025, grow for another hour (OD back to 0.1), and then add transforming DNA.  

The postdoc is making me a prep of NovR PCR fragment that I can use for these transformations.  I'll want to do a test transformation first, using this DNA with normal competent cells, to determine how much DNA to use.  We don't want to use MAP7 chromosomal DNA because (1) the efficiency is low because most of the DNA is from other parts of the chromosome, and (2) it will transform other parts of the competent cells' chromosomes, potentially removing the mutation we want to isolate.

Incubate the cells with DNA for 30 min, add DNaseI and wash the cells by filtration to remove both the DNA and dead cells.  Resuspend in medium with novobiocin and grow for at least 6 hr (or overnight) before plating on nov5 plates. (We usually use nov at 2.5 µg/ml, but NovR transformants grow just as well on plates with 5 µg/ml, and most spontaneous NovR mutants don't.)  Freeze some of the cells instead of plating them all.

If the cultures are grown overnight with novobiocin,  I should probably increase the novobiocin to 5 µg/ml after the first few hours.  In the morning I could plate some of the cells for NovR colonies, and just grow some in log phase for 2-3 hr before transforming them with KanR DNA to select the ones that are genetically hypercompetent (eliminating the ones that were accidentally competent for the first selection).  Alternatively I can wait and pool the NovR colonies that grow up on the plates.  Because the culture should be already enriched for hypercompetent mutants I won't need to worry about 'bald spot' effects but can plate relatively dilute cultures on the kan plates.  (I won't assume this but check by plating different concentrations of course.)

Next I pool all the transformants from each StrR or CmR culture (KanR if I've done the second round of selection, NovR if I haven't) and prep DNA from them.  I use the DNA to transform competent wildtype cells to StrR or CmR, grow the pooled cells into log phase, and select for hypercompetent cells with NovR DNA.  (This could be a PCR fragment or chromosomal DNA (wait, do I have a NalR strain in the freezer?).)

For the wildtype culture, I can just do one more round of hypercompetence selection, with NalR, or I can do an unselected transformation (or select for NovR as an unlinked marker) and then do the NalR hypercompetence selection.

Pool the NalR transformants from each culture, extract DNA and sequence.  If there are hypercompetent mutants we expect to see peaks of novel alleles at and around the sites of the mutations.

So, the whole experiment:  First streak out the cells.  Then dilute and grow, mutagenize, wash, dilute and grow, freeze, dilute and grow, transform with NovR, wash, grow with nov, freeze, grow with nov, plate.  (Pool), dilute and grow, transform with KanR, plate.  Pool KanR, make DNA, transform to CmR or StrR, plate.  Pool, dilute and grow, transform to NalR, plate, pool, make DNA, sequence.

AAACCKKK!  Maybe it will be clearer if I partition it into days?

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

Well, at least I now know what I'll be getting into.

Selecting for rare NovR cells by enrichment in broth - would it work?

In the previous post about searching for new hypercompetent mutants I mentioned that our ability to find rare NovR transformants in a log-phase culture is limited by the need to put relatively small numbers of NovS cells (less than 5 x 10^7) on each plate.  If we use more cells we see large 'bald spots' where the NovR cells are unable to form colonies; we speculate that this is because of toxic effects of too many NovS cells dying around them.  When the problem is severe we see no NovR colonies at all even though hundreds were plated.

One solution is just to distribute the cells over more and bigger plates.  Scaling up by ten-fold is easy, but scaling up by 100-fold is a lot more work and more expense for plates and medium.  In the experiment we're now considering, we expect the NovS cells to outnumber the NovR cells by about 10^8 to 10^9, so we'd like to scale up by a thousandfold.

An alternative that we've never tried is to add novobiocin to the liquid culture for a few hours before plating the cells on agar medium containing novobiocin.  This scaling up would let us amplify the rare NovR cells by letting them double repeatedly while the NovS cells stalled or died.  Ten doublings (about 5 hr of growth) would bring the NovR density up from 10^-9 to 10^-6, so that plating on a moderate number of plates would capture the full diversity of the initial transformant population.  We would no longer be able to assume that separate NovR colonies descended from independent transformations, but for this experiment that's not important.

I think I'll try this today, using a normal log-phase culture rather that the EMS-mutagenized cells we'd use in the planned mutant-hunt:

1. Start with a mixture of competent cells and log-phase cells, such that I expect a transformation frequency of about 10^-7.  This frequency is higher than we would see in the planned mutant-hunt, because I want to have a predictable and easily measured number of transformants to start with.  
2. Incubate 5 ml cells at a density of 10^8 (OD600 = 0.03) with 1 µg MAP7 DNA for 15 min,  Plate an aliquot with and without novobiocin.  Dlilute the rest of the culture 100-fold with fresh medium and let grow for 1 hr, and then add novobiocin at 2 µg/ml to prevent further growth of NovS cells.
3. Plate aliquots again and at hourly intervals to check on the growth and survival of the NovS and NovR cells.
4. Just in case the dying NovS cells are toxic in the liquid culture, though they'll be much more dilute than on plates, after a couple of hours of novobiocin selection I'll wash the culture by filtration and resuspend the cells in fresh novobiocin medium and let them continue growing.  This will only take a few minutes and will also remove the residual NovR DNA.
5. After five hours of selection the NovR cells will have doubled about 10 times.  If the NovS cells survived growth in novobiocin but didn't divide I should see a 1000-fold increase in the frequency of NovR colonies on my plates, from about 10^-7 to about 10^-3.  If most or all of the NovS cells died the increase will be even greater.

If this works as expected, we'll be able to start our big experiment with a very large mutagenized culture and pool many more independent transformants.  This will give us a much more diverse pool of hypercompetent mutants for our sequencing.

 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

OK, the first try didn't work because I appear to have added bacteria to the sBHI agar instead of novobiocin (!!!) so what should have been novobiocin plates were plates with about 10^4 tiny colonies of H. influenzae embedded in the agar!

But the second try worked very nicely.  The red lines are the planned experiment, with a small number of competent cells added to a 100-fold excess of non-competent cells; the green lines are just the competent cells by themselves.  Each cell prep was incubated with NovR transforming DNA for 15 minutes, and then novobiocin was immediately added to the culture.  The non-competent cells (open red squares) continued growing a bit and then stalled (the competent cells had already stopped growing because they had been starved to induce competence).  After two hours the transformed NovR cells (solid red and green squares) started doubling and the non-transformed NovS cells (open red and green squares) started dying, just as they should.  By the next morning the cultures were thick and consisted of only NovR cells.

This result means that we can incorporate selection for NovR transformants in broth in the mutagenesis experiment and so won't  have to distribute the transformation mixture over hundreds of Nov plates.

A better strategy for finding new hypercompetence mutations in murE

If we did decide to buy some EMS (it's cheap and readily available from Sigma) and repeat the mutagenesis, I think we should use a different strategy to select for hypercometent mutants.  Specifically, we should focus on getting more  strains whose hypercompetence is caused by mutations in murE (see this old post for a description of the murE results).  We have four such mutants now, which change two different amino acids, and we don't understand how they work.  Having more independent mutants would help clarify the situation, whether we get new mutations or just more of the same mutants.

How to do this?  We would start by mutagenizing cells with EMS, but instead of using wildtype cells we'd use cells carrying a neutral chloramphenicol-resistance insertion (CmR) that's within a few kb of murE.  The mutagenized cells would be incubated in log phase with a NovR DNA fragment, and the rare transformants wuld be selected by plating on novobiocin and pooled.  Since the transformation frequency of wildtype cells in log phase is less than 10^-9, many of these should be hypercompetent mutants.  DNA from the pooled transformants would be used to transform wildtype cells, selecting for CmR to enrich for mutations in and near murE, and these 'backcrossed' transformants would again be selected for hypercompetence by transformation in log phase, this time using MAP7 DNA and selecting for streptomycin resistance.  DNA from the pooled transformants would be sequenced, as would individual hypercompetent isolates.

We need to think about the numbers.  Say 1-5% of the original NovR transformants are hypercompetent strains with murE mutations (in the original experiment we found 4/150).  If we pool their DNA and backcross, selecting first for CmR, 1-5% of the colonies will have murE hypercompetence mutations.  Even if these mutants were only 100-fold more competent in log phase than normal cells, selecting for these by transformation to StrR would give mostly colonies with the desired mutations.

(The murE mutants we have are about 10^6-fold more competent, so we might miss mutations giving weaker hypercompetence phenotypes...)

Timing:

1. Day 1.:Streak out the CmR strain.
2. Day 2: Grow the CmR strain and mutagenize for 30 min with EMS.  Wash away the EMS, freeze some of the cells for later work. Dilute the rest right away and grow in log phase for 2-3 hr or more.  Add NovR DNA for 30 min and plate on lots of plates.  
3. Day 3: At this stage each hypercompetent NovR colony is likely to be an independent mutant.  Pool all of them and isolate DNA.
4. Additional Day 2?: We want many hundreds of transformants, and can scale up the mutagenesis and transformation cultures,  but the selection is limited by the need to not put more than ~5x10^7 NovS cells on each plate.  Maybe we should also try enriching for NovR before plating, by adding novobiocin to the broth and growing for a few hours or overnight.  This would let us screen a lot more cells and thus find more mutants, although we'd sacrifice independence.  Again we'd then isolate DNA from the NovR culture or pooled NovR colonies.
5. Still Day 3: Transform competent wildtype cells with this DNA, selecting for CmR.   The expected transformation frequency is about 10^-4 to 10^-3, so we can easily select many thousands of independent transformants to pool.  1-5% of these should have the desired hypercompetence mutations.
6. Day 4: Grow the pooled transformants and transform them in log phase to StrR.  
7. Day 5: Almost all the transformants should be the desired hypercompetence mutants. Pick some of these for competence testing and pool the rest.
8. Prep the DNA of a number of individual isolates and of a large pool of colonies.  Sequence these.  Look for specific mutations in the individual cultures and for enrichment of mutations in the pool.

Should we also do an unfocused search for any hypercompetence mutations?  We could do this in parallel, replacing the selection for CmR on Day 3 with selection for NovR.  We might still get mainly mutations in murE, because these give such a strong phenotype.  If we wanted to target mutations in sxy, we could first do a second round of selection for transformation using StrR MAP7 DNA and then select for StrR in the backcross.  Since StrR is not very close to sxy we'd want to be gentle with our Day 3 DNA prep so the fragments were long.

A new way to make money from researchers?

Basically, you give World Biomedical Frontiers $38 and they list your paper's Abstract on their website along with whatever supplementary explanation of the work you provide.  I gather that "cutting-edge biomedical research" means "research by people who gave us $38".

A bit of surfung suggests that you might then add a note like this to your publication list:

"This paper has been selected to be featured in World Biomedical Frontiers (http://biomedfrontiers.org/cancer-2013-may-2-5/) because of its innovation and potential for significant impact. World Biomedical Frontiers [ISSN: 2328-0166] focuses on cutting-edge biomedical research from around the globe."

(We don't work on influenza.)

Dear Dr. Redfield:

Your recent paper about influenza-“Defining the DNA uptake specificity of naturally competent Haemophilus influenzae cells” (published in “Nucleic acids research.2012 Sep;40(17):8536-8549.”) has been selected to be featured in our next issue of World Biomedical Frontiers, because of its innovation and potential for significant impact.

Research results with significant potential to improve health – or to treat or prevent disease – often deserve an immediate leap onto the “front page”. However, scientific breakthroughs don't always make the front page – and some don't make any page! We are the platform for you to stand out from among ~100,000 papers published each month, in order to attract more attention from the public and potential investors.

World Biomedical Frontiers [ISSN: 2328-0166] focuses on cutting-edge biomedical research from around the globe. Our website receives more than 11,000 visits per month from an international audience of academic and industrial researchers and developers, providing greater opportunity for your results to be recognized and appreciated.

If you accept our invitation to feature your paper on our website, a $38 processing fee will be charged. We will then post the abstract/summary of your paper in the latest section of Infection and Immunity, with additional information from you highly recommended to further explain your novel findings and concepts in plain language; photos and/or figures are welcomed. Here are two examples (1 and 2).

In order to report breaking publications in a timely fashion, we ask that you contact us within 2 weeks if you wish your paper to be featured in our next issue.

Sincerely,

Michael S. Yang, M.D & Ph.D.
Editor
World Biomedical Frontiers, LLC
New York, USA
Phone: 1-(917) 426-1571
E-mails: frontiers@biomedfrontiers.org
Website: http://biomedfrontiers.org/